Th overall objectives of this proposal are 1) to continue the development of new methods and DNA probes for the analysis of human chromosomes and genes in normal and neoplastic cells or tissues, 2) the implementation of multiparameter fluorescence imaging technology that can be applied to the simultaneous analysis of multiple chromosomes, genes, mRNAs or proteins in diverse biological specimens, and 3) the examination of genetic changes occurring in squamous cell carcinomas of the head and neck. Specific research goals are: 1. to develop an automated method of chromosome karyotyping based on multicolor fluorescence in situ hybridization (FISH). Twenty-four chromosome-specific "painting" probe libaries and an Alu-PCR "banding" probe will be hybridized simultaneously to chromosome preparatuibs, Each probe set will be labeled with one or more of five spectrally distinct fluors which, when used in simple boolean combination, will discriminate all 25 probes. The digital imaging microscope will have all stage scanning operations, optical filter changes, source fluorescence image capture, CCD data downloading and identification of probes by spectral signature under computer control Cell lines containing cytogenetically defined chromosomal abnormalities will be used, in a blind fashion, to test, refine and compare FISH karyotyping with conventional cytogenetics. Once validated, multicolor FISH will be used to karyotype chromosome preparations from short-term cultures of ovarian and breast tumor tissues obtained by needle aspiration and biopsy. 2. to evaluate and optimize the multiplex analysis of chromosomes, genes, mRNA and proteins in cultured cells and fresh frozen or formaldehyde-fixed, paraffin-embedded tissues by determining how many probes can be analyzed simultanelusly and resolved unequiocally by quantitative multicolor fluorescence microscopy, and to establish the extent to which this is influenced by probe size, gnetic complexity of the probe or the copy nmber of the target analytes. 3. to identify significant genetic anomalies in squamous cell carcinomas of the head and neck by determining gene dosage abnormalities (aneuploidy, loss of heterozygosity or gene amplification) and by comparing the genotypic anomalies found in individuals with multiple primary carcinomas with those present in individuals with single primary lesions. Genetic analysis will be done by a combination of comparative genome hybridization (CGH), FISH, loss of heterozygosity (LOH) studies using polymorphic microsatellite markers amplified by PCR, and analysis of potential expensions of nucleotide repeats. Slection of tumor specimens from the YALE tissue bank and evaluation of genotypic data for clinical correlates with tumor staging, patient survival or occurrence of metastatic disease will be done in collaboration wit institutional oncologists and pathologists.